Rapid gut growth but persistent delay in digestive function in the postnatal period of preterm pigs

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1 Am J Physiol Gstrointest Liver Physiol 31: G55 G56, 216. First pulished Jnury 28, 216; doi:1.1152/jpgi Rpid gut growth ut persistent dely in digestive funtion in the postntl period of preterm pigs Crl Frederik Hnsen, 1 Thoms Thymnn, 1 Anders Dniel Andersen, 1 Jens Juul Holst, 2 Bolette Hrtmnn, 2 Lind Hilsted, 4 Louise Lnghorn, 1 Jo Jelsing, 3 nd Per Torp Sngild 1,5 1 Comprtive Peditris nd Nutrition, University of Copenhgen, Copenhgen, Denmrk; 2 Novo Nordisk Foundtion Center for Bsi Metoli Reserh, nd Deprtment of Biomedil Sienes, Fulty of Helth nd Medil Sienes, University of Copenhgen, Copenhgen, Denmrk; 3 Gur, Hørsholm, Denmrk; 4 Deprtment of Clinil Biohemistry, Copenhgen University Hospitl, Cophenhgen Denmrk; nd 5 Deprtment of Peditris nd Adolesent Mediine, Copenhgen University Hospitl, Denmrk Sumitted 6 July 215; epted in finl form 22 Jnury 216 Hnsen CF, Thymnn T, Andersen AD, Holst JJ, Hrtmnn B, Hilsted L, Lnghorn L, Jelsing J, Sngild PT. Rpid gut growth ut persistent dely in digestive funtion in the postntl period of preterm pigs. Am J Physiol Gstrointest Liver Physiol 31: G55 G56, 216. First pulished Jnury 28, 216; doi:1.1152/jpgi infnts often tolerte full enterl nutrition few weeks fter irth ut it is not known how this is relted to gut mturtion. Using pigs s models, we hypothesized tht intestinl struture nd digestive funtion re similr in preterm nd term individuls t 3 4 wk fter irth nd tht erly enterl nutrition promotes mturtion. or term esrendelivered pigs were fed totl prenterl nutrition, or prtil enterl nutrition [Enterl (Ent), ml kg 1 dy 1 of ovine olostrum] for 5 dys, followed y full enterl milk feeding until dy 26. The intestine ws olleted for histologil nd iohemil nlyses t dys, 5, nd 26 (n 8 12 in eh of 1 tretment groups). Intestinl weight (reltive to ody weight) ws redued in preterm pigs t 5 dys ut ENT feeding stimulted the muosl volume nd peptidse tivities. Reltive to term pigs, muosl volume remined redued in preterm pigs until 26 dys lthough plsm glugon-like peptide 2 (GLP-2) nd gluose-dependent insulin-trophi peptide (GIP) levels were inresed. pigs lso showed redued hexose sorptive pity nd rush-order enzyme (surse, mltse) tivities t 26 dys, reltive to term pigs. Intestinl struture shows remrkle growth dpttion in the first week fter preterm irth, espeilly with enterl nutrition, wheres some digestive funtions remin immture until t lest 3 4 wk. It is importnt to identify feeding regimens tht stimulte intestinl mturtion in the postntl period of preterm infnts euse some intestinl funtions my show long-term developmentl dely. premturity; gut development; enzymes; digestion; glugon-like peptide 2 ABOUT 1% OF ALL INFANTS re orn preterm ( 37 weeks of gesttion) nd the proportion is inresing in most ountries (6). infnts show n inresed suseptiility to infetions, respirtory distress syndrome, intrrnil hemorrhge, ronhopulmonry dysplsi, retinopthy of premturity, extruterine growth restrition, neurodevelopmentl disturnes, nd nerotizing enteroolitis (NEC) (21, 31). The hllenges re inversely relted to gesttionl ge t irth nd Address for reprint requests nd other orrespondene: P. T. Sngild, Comprtive Peditris nd Nutrition, Dept. of Clinil Veterinry Mediine nd Animl Siene, Fulty of Helth nd Medil Sienes, 68 Dyrlægevej, DK-187 Frederikserg, C, Denmrk. the most severe dpttion prolems our during the first few weeks fter irth. If preterm infnts survive the diffiult neontl period, their ody funtions often dpt well, lthough some orgn systems my show long-term developmentl dely or defiits (18). It remins unknown how different orgn systems dpt in preterm infnts nd when prmeters of struture nd funtion eome similr to those in term infnts. For gstrointestinl (GI) funtions, eting disorders nd dysregulted ppetite hve een reported in preterm infnts (44) ut very little is known out the long-term dpttion of the GI trt in preterm neontes. Investigtions of postntl GI dpttion in preterm neontes re diffiult without n niml model tht omines preterm irth, high NEC risk, nd linilly relevnt feeding interventions, suh s prenterl nd enterl nutrition. Piglets delivered t 9% gesttion suffer from mny of the sme disorders nd physiologil prolems s modertely immture humn infnts nd they hve een widely used to investigte the immedite feeding-, diet-, nd miroiot-relted gut omplitions (39, 41, 43). The timing of perintl GI mturtion ppers to e intermedite in pigs, reltive to the delyed nd rpid postntl GI development in rodents, nd the reltively erly (prtly prentl) nd slower development in humns (39). On the other hnd, the high NEC sensitivity in preterm pigs, even fter moderte (1%) redution in gesttionl length, suggests tht the piglet GI trt is reltively immture t irth. This is n dvntge when ttempting to model spets of GI development in very immture infnts. Using this model, we hve doumented tht NEC sensitivity is highly diet dependent nd tht porine, ovine, nd humn intt milk diets protet ginst NEC, reltive to infnt formuls (5, 26). Animl studies indite tht erly enterl feeding is importnt for gut mturtion nd prolonged totl prenterl nutrition (TPN) indues intestinl trophy nd dysfuntion (8). Erly GI mturtion my help to prevent lter dverse effets of slow growth nd indequte nutrient intke in preterm infnts (3). On the other hnd, too rpid dvnement of enterl feeding is ssoited with high NEC risk, nd delyed dvnement of enterl feeding my derese NEC risk in oth preterm infnts nd pigs (2, 32, 33). Consequently, grdul introdution of mother s own milk, donor milk, or infnt formul ( miniml enterl nutrition, dily inreses of 1 2 ml kg 1 dy 1 ), is often used for 1 2 wk fter irth s djunt to prenterl nutrition (PN), efore trnsition to full enterl feeding t 2 4 wk (e.g., ml kg 1 dy 1 ). Regrdless, the optiml feeding time, dvnement rte, nd diet (espeilly when G /16 Copyright 216 the Amerin Physiologil Soiety

2 mother s milk is not ville) remin unler. It is lso unknown whether enterl feeding during the first week my hve oth short- nd long-term onsequenes for GI mturtion. The GI trt not only filittes nutrient digestion nd sorption ut hs mjor endorinologil, neurologil, nd immunologil funtions tht re ruil for long-term helth. Three gut hormones relesed in response to feeding nd with effets on gut growth nd funtion re gstrin relesed from gstri G ells, gluose-dependent insulin-trophi hormone (GIP) from (proximl) intestinl K ells nd glugon-like peptide 2 (GLP-2) from (distl) intestinl L ells. infnts show elevted levels of these GI hormones, ompred with term infnts nd dults, nd they re sensitive to enterl stimuli (1, 16, 27). During totl prenterl nutrition (TPN), in the sene of enterl food, dministrtion of the gut tropi hormone, GLP-2, mrkedly improves gut growth nd dpttion in preterm nd term pigs (9, 36, 38), ut the role of this nd other GI hormones on lter GI development remins unknown. In the present study, we hypothesized tht intestinl struture nd digestive funtion re redued y preterm irth ut tht gut mturtion our postntlly so tht the preterm gut rehes stge of mturity t 3 4 wk tht is similr to tht in term nimls. Furthermore, we hypothesized tht smll mounts of enterl food (vs. TPN) during the first week of life would ffet gut mturtion oth within the first week nd lter (e.g., t 3 4 wk). Bovine olostrum ws used s the first enterl diet euse it is known to protet ginst NEC in piglets (9, 36, 38) nd hs een tested s the first enterl diet in preliminry studies in preterm infnts (28). Intestinl morphology nd muos volume were mesured together with key mrkers of digestive (e.g., hydrolse tivities, nutrient sorption) nd enteroendorine ell funtions (plsm gstrin, GLP-2, nd GIP levels). The results help to understnd how the immture GI trt mtures fter preterm irth until 3 4 wk of ge when oth preterm pigs nd modertely preterm infnts tolerte full enterl feeding nd re linilly stle enough to leve their intensive re units. METHODS Animls nd nutrition. One hundred nd sixty-eight piglets from eight sows (Dnish Lndre Lrge White Duro) were esren delivered, either preterm (dy 16 or 9% of gesttion; n 112 pigs from five sows) or t full term (dy 118 or 1% gesttion, n 56 pigs from three sows) ording to preestlished protools (43). All piglets were immeditely trnsferred to piglet neontl intensive re unit nd rered in temperture-, moisture-, nd oxygen-regulted inutors. All preterm nd term pigs were fitted with orogstri (6 Fr; Phrmplst, Roskilde, Denmrk) nd umilil rteril theters (4 Fr; Portex, Kent, UK) nd lok-rndomized sed on irth weight in two groups reeiving totl prenterl nutrition (TPN) or PN plus miniml enterl nutrition with ovine olostrum (ENT) for the first 5 dys. Within the first 24 h they reeived 12 ml/kg of their mother s plsm intr-rterilly to otin immunologil protetion. Throughout the 5 dys of theteriztion we provided ontinuous infusion of heprin vi the PN solution to void thromosis. For oth preterm nd term TPN groups, PN (modified omintion of Kiven, Vitlipid, Soluvit, nd Vmin; Fresenius Ki, Bd Homurg, Germny) (43) ws given vi umilil theteriztion t 96 ml kg 1 dy 1 on dy 1, grdully inresing to 144 ml kg 1 dy 1 on dy 5 when the theters were removed from ll nimls. The nutrient omposition of the TPN ws (in g/l) rohydrtes (71.4), mino ids (44.6), nd ft (3.9) with totl energy density of 745 kl/l. For the ENT group, enterl nutrition with ovine olostrum (BC) ws provided s solution of 17 g olostrum powder, otined from Biofier Dmino, Vejen, Denmrk, mixed in 1 liter of sterile wter. The BC ws given in oluses every 3 h, strting t 16 ml kg 1 dy 1 on dy 1 nd inresing to 64 ml kg 1 dy 1 on dy 5. The ENT group ws provided with supplementl PN suh tht the two dietry regimens were isoenergeti (74 11 kl kg 1 dy 1 over the first 5 dys) s desried in previous study using the sme initil diet regimen (45). On dy 5, the PN ws disontinued nd ll piglets were given inresing mounts of rw ovine milk (64 15 ml kg 1 dy 1 )tody 9 nd susequently trnsferred to reonstituted whole milk powder t 15 2 ml kg 1 dy 1 (Arl Foods Ingredients, Viy, Denmrk) until dy 26. The dosing strtegies for oth TPN nd enterl feeding re omprle with the feeding dvnement rtes used for modertely preterm infnts (4, 13, 17, 25, 53). See Fig. 1. To prevent infetions nd sepsis, moxiillin trihydrte (Prillin 7%, MSD, Animl Helth, Bllerup, Denmrk) ws dministered prophyltilly in the feed (2 mg/l) on dys Animls were euthnized with pentoritl (5) nd tissues olleted t dys, 5, nd 26, resulting in totl of 1 tretment groups (neworn dys, 5-dy-old TPN nd ENT, 26-dy-old TPN nd ENT fter either preterm or term delivery). All niml experiments were pproved y the Dnish Committee for Animl Reserh (liense no ). Plsm hormone mesurements. Plsm smples for gstrin, GLP-2, nd GIP mesurements were olleted t euthnsi. For gstrin, smples were nlyzed s desried previously, with n ntiody vlidted for pigs (ode no. 264) (4). For GLP-2, smples were extrted in finl onentrtion of 75% ethnol nd GLP-2 ws mesured with n NH 2-terminl speifi ntiserum (ode no. 9216), mesuring only GLP-2 with n intt NH 2 terminus, s previously desried (24). For stndrds, reominnt humn GLP-2 nd rt GLP-2 trer with n Asp 33 Tyr 33 sustitution ws used. After plsm extrtion (7% ethnol), totl GIP mesurements were performed y using n ntiserum reognizing the COOH-terminl prt (ode no. 8867), s previously desried (29). Ltulose-mnnitol test. To mesure in vivo intestinl permeility, piglets were dosed intrgstrilly vi n orogstri tue with omined 5% ltulose nd 5% mnnitol solution. A olus of 15 ml/kg ody wt ws given 3 h efore euthnsi fter withdrwl of feeds for 3 h. At the time of euthnsi, urine ws olleted y intr-dominl ystoentesis. The urine smples were stored t 2 C nd nlyzed spetrophotometrilly y n end-point ssy, s previously desried (5, 7, 34). Dy - 5 Dy 6-26 ENT TPN ENT TPN Bovine Milk G551 Fig. 1. Study outline. Animls were esren delivered, either preterm (9% gesttion) or t full term, nd rndomized into 1 of 2 groups: totl prenterl nutrition (TPN) or prenterl nutrition plus miniml enterl nutrition with ovine olostrum (ENT) for the first 5 dys. On dy 5, the prenterl nutrition ws disontinued nd ll piglets were given inresing mounts of ovine milk until dy 26.

3 G552 Gltose test. The test ws performed y dministering 15 ml/kg orl olus of 5% gltose solution vi n orogstri tue 2 3 h fter the lst olus feeding on dy 4 or 25, respetively. Blood smples were olleted either vi the umilil theter, or vi jugulr vein punture, t or 2 min fter the gltose olus. Plsm ws isolted nd nlysis of gltose onentrtion ws performed s previously desried (48). Tissue nd lood smpling. Following indution of nesthesi, lood ws drwn y rdi punture nd the pigs were then euthnized with pentoritl (5). The finl lood smple ws tken 2 4 h fter irth for neworn pigs nd extly 1.5 h fter the lst mel for the 5- nd 26-dy-old pigs. Blood smples were entrifuged nd the plsm frtion ws rpidly frozen. The smll intestine, from the pylori sphinter to the eum, ws rpidly exised nd pled on n ie-old metl plte in relxed stte. The totl length nd weight were mesured, nd the intestine ws smpled for histology nd stereology with systemti uniform rndom smpling. A totl of nine setions were tken throughout the intestine strting from predetermined rndom strt site nd with set smpling distne (totl length/9). At eh site, 15-mm trnsverse iopsy ws tken nd fixed in 4% neutrl uffered prformldehyde. Ex vivo rush order enzyme tivities. Ativities of rush order enzymes, ltse, mltse, surse, minopeptidse N (ApN), minopeptidse A (ApA), nd dipeptidyl peptidse 4 (DPP4) were nlyzed in homogentes of proximl, middle, nd distl intestinl tissues y spetrophotometry, s desried previously (42). Enzyme tivities were expressed s units per grm of wet tissue. Tissue homogentes were otined y homogenizing the tissue smple in 1% Triton X-1 wter solution (1 ml/g tissue). Enteroendorine ells deteted y immunohistohemistry. Enteroendorine ells were identified y immunohistohemistry using the following proedure. Setions were deprffinized in xylene nd rehydrted. Setions were then sujeted to ntigen retrievl in Tris- EGTA uffer nd loked for endogenous peroxidse tivity nd nonspeifi inding, efore eing inuted with GLP-1 ntiody diluted 1:16, (GLP-1F5 P9, gift from Novo Nordi, Copenhgen, Denmrk) overnight. Setions were susequently visulized with Ultrvision One (TL-15-HDJ Thermo Sientifi) nd finlly developed y using DAB s hromogen. Slides were ounterstined with hemtoxylin, dehydrted, overslipped with Pertex (Skur), nd finlly digitlized on n Aperio Snsope AT slide snner (Aperio). The loliztion nd shpe of the positively stined ells onfirmed tht these were indeed enteroendorine ells. The sme ntiody hs previously een vlidted for visulizing GLP-1-positive L ells in pigs (3). Muosl struture nd stereologil estimtion of muos volume. To evlute muosl morphology, fixed smples from the proximl, middle, nd distl regions of the smll intestine were emedded in prffin, setioned, nd stined with hemtoxylin nd eosin efore mesurement of villus height nd rypt depth with light mirosopy imges vi the ImgeJ proessing nd nlysis softwre progrm, s desried previously (23). The intestinl muos volumes were estimted y using newcast (Visiophrm) on digitl slides. For eh of the nine intestinl setions, fields of views were smpled in rndom systemti mnner y use of the newcast softwre. The volumes of the muosl lyer, s well s the sumuos/musulris/seros lyer, were estimted y use of 16-point grid t 1 mgnifition. The numer of points hitting the struture of interest ws onverted into volume y using the priniple of Cvlieri: Vol ref p A(p) t, where p is the totl numer of points hitting the struture of interest, A(p) is the re ssoited with eh grid point, nd t is the distne etween setions (23). Estimtion of the totl numer of enteroendorine ells. The totl numer of immunoretive GLP-1-positive L ells in the smll intestine ws estimted y the priniple of the physil dissetor (22, 47). For this purpose 5- m-thik tissue setions were smpled s two onseutive levels. The slides were proessed for immunoretivity, digitized, nd finlly nlyzed on omputer running newcast (Visiophrm) softwre t 2 mgnifition. The totl numer of stined ells in defined smpling volume ws ounted nd the prtile density N v ws lulted s N v [ Q/(frme)] h p, where Q is the totl numer of uniquely ounted ells, (frme) is the re of the ounting frme, h is the distne etween the two setions, nd p is the totl numer of points hitting the referene spe. The totl numer of enteroendorine ells ws finlly determined y multiplying N v with the totl referene volume. Sttistis nd dt presenttion. Results re presented s mens SE unless otherwise stted. All dt were nlyzed with pig nd litter s rndom vriles nd time, tretment nd intestinl region s fixed vriles in the mixed proedure of the SAS sttistil softwre progrm (SAS version 4.3; SAS Institute, Cry, NC). Post ho omprison ws rried out without orretion nd only within sme gesttionl ge (preterm or term). Speifilly for enzymti tivities, tretment omprisons (ENT, TPN) were rried out for eh intestinl region (proximl, mid, distl) t eh of the postntl ges (, 5, 26 dys). Proility levels elow.5 were onsidered signifint. RESULTS Body weight gin nd linil ondition. Reltive to term, preterm piglets hd lower ody weight t irth (P.6), with the differene inresing further to 5 dys (P.1) nd 26 dys (P.1, Fig. 2A). This led to n overll redued ody weight gin in preterm vs. term pigs ( vs g kg 1 dy 1, P.1), despite their identil nutrient intkes per kilogrm ody weight. The slower growth my e prtly explined y lowered nutrient digestion in the preterm pigs sine these showed more dys with dirrhe, reltive to term piglets (P.1, s nlyzed ross diet groups, Fig. 2B). TPN-fed preterm pigs displyed the highest numer of dys with dirrhe nd ENT term pigs the fewest dys with dirrhe (Fig. 2B). A detiled desription of the linil ondition, lood hemistry vlues, orgn weights, ody omposition, nd ehviorl hrteristis of - to 26-dy-old preterm nd term pigs is pulished s seprte report (2). Gut morphology, muos volume, nd gut weight. At ll three ges, preterm pigs showed lowered solute vlues for intestinl length (P.1, Fig. 2C), intestinl weight (P.5, dt not shown), nd intestinl muos volume (P.5, Fig. 2D), ompred with term pigs. When expressed reltive to ody weight, the intestinl weight nd muos volume were signifintly lower in preterm pigs, ut only t irth (P.1, dt not shown). At dy 5, oth term nd preterm ENT nimls displyed higher men muos volume, ompred with TPN, ut the inrese did not reh sttistil signifine. On the other hnd, the tendeny to inresed solute intestinl weight in the 5-dy-old ENT pigs (Fig. 2D) eme highly signifint when expressed reltive to ody weight ( vs g/kg, P.1 for ENT vs. TPN ross preterm nd term pigs). Villus height nd rypt depth. Villus height tended to derese with ge for oth preterm nd term nimls nd were generlly lower for preterm thn for term pigs (P.1, Fig. 3A). With ge, rypt depths inresed mrkedly for oth preterm nd term nimls (Fig. 3, B nd D), likely refleting inresed mitoti tivity with dvning ge (P.5, Fig. 3C). Aross gut regions nd gesttionl ges, ENT feeding tended to lower the villus/rypt rtio, reltive to TPN (Fig. 3C, P.7), ut the differenes within eh postntl ge group

4 Body weight (g) A Age (d): B Men numer (dys) Body weight ENTTPN ENTTPN ENT TPN ENTTPN Dys with dirrhe ** * C Smll intestinl length (m) Volume (m 3 ) Age (d): D Intestinl length ENT TPN ENT TPN ENT TPN ENT TPN Muos volume * ** G553 ENT TPN ENT TPN Age (d): ENT TPN ENT TPN ENT TPN ENT TPN Fig. 2. Body weight (A), umultive dys with dirrhe (B), intestinl length (C), nd muosl volume (D) in -, 5-, nd 26-dy-old preterm or term pigs fed totl prenterl nutrition (TPN) or supplemented with enterl nutrition (ENT) for the first 5 dys (mens SE). Columns within the preterm or term groups not shring the sme letter re signifintly different (P.5). *P.5, **P.1, P.1, signifint differenes etween preterm nd term piglets for eh postntl ge group (, 5, 26 dys), nlyzed ross diets (ENT, TPN). d, Dys. remined mrginl. Figure 3D shows representtive mirogrphs of setions from the middle intestine of preterm nd term pigs t the three different ges. The pnels indite the deresing villus height per rypt depth rtio with dvning postntl ge, reltively open villus struture in preterm neworn pigs (Fig. 3D, top left), nd muh higher density of immture vuolted ells in preterm 5-dy-old pigs (open white res on villus), reltive to the term 5-dy-old pigs (rrows in Fig. 3D, middle). Gut peptide levels in plsm nd the numer of enteroendorine ells in the smll intestine. Gstrin, GLP-2, nd GIP plsm levels were mesured in ll pigs t the time of tissue olletion, i.e., 2 4 h fter irth for neworn pigs nd 1.5 h fter the lst mel for 5- nd 26-dy-old pigs. At irth, plsm gstrin levels were signifintly higher in preterm pigs, reltive to vlues in term pigs (63 7 vs pmol/l, P.1), ut t 5 nd 26 dys vlues were similr ross ll delivery nd diet groups (2 3 pmol/l, dt not shown). Only t 5 dys ws there tendeny to inresed vlues in ENT vs. TPN pigs (26 3 vs. 2 2 pmol/l ross delivery groups, P.9). Anlyzed ross ll postntl ge groups, GLP-2, sereted minly from L ells in the distl intestine, showed elevted levels in plsm of preterm vs. term nimls, most lerly t 26 dys (P.5, Fig. 4A). The ENT tretment stimulted plsm GLP-2 levels t 5 dys in term pigs ( 6% inrese, P.5) wheres the effet in preterm pigs ws less pronouned (P.11, 3% inrese). For oth groups, the levels inresed with ge (P.1), with the highest GLP-2 levels deteted in 26-dy-old preterm pigs fed ENT from irth (Fig. 4A). GIP, sereted minly y proximlly loted K ells, lso inresed in onentrtion in plsm with dvning ge (P.1 nlyzed ross preterm nd term pigs, Fig. 4B). The ENT tretment ws ssoited with very mrked inrese in preterm pigs ( 2% inrese, P.1) wheres the effet in term pigs ws less pronouned ( 5% inrese, P.7). Importntly, these

5 G554 Villus length A 4 Dy Villus length (µm) 6 D 2 B Crypt depth 1 Dy 5 Crypt depth (µm) 15 5 C Villus/rypt rtio ** 1 Dy 26 * ** 5 ENT TPN ENT TPN Age (d): 5 26 ENT TPN 5 ENT TPN 26 Fig. 3. Villus height (A), rypt depth (B), nd villus/rypt rtio (C) in -, 5- nd 26-dy-old preterm or term pigs fed totl prenterl nutrition (TPN) or supplemented with enterl nutrition (ENT) for the first 5 dys (mens SE). Representtive photomirogrphs (D) tht indite intestinl morphology in term nd preterm nimls throughout the study period re shown for the middle intestine of pigs supplemented with ENT. Arrows indite immture vuolted ells in the preterm intestine. Columns within the preterm or term groups not shring the sme letter re signifintly different (P.5). *P.5, **P.1, P.1, signifint differenes etween preterm nd term piglets for eh postntl ge group (, 5, 26 dys), nlyzed ross diets (ENT, TPN). effets were trnsient sine they were no longer evident t dy 26 (Fig. 4B). To speifilly investigte whether the high plsm GLP-2 levels in ENT 26-dy-old pigs (Fig. 4A) refleted higher numer of L ells, their numer nd density ws estimted y stereologil methods in the preterm group of nimls t dys nd 26 following ENT nd TPN nutrition. The numer of GLP-1 immunoretive L ells were distriuted ross the rypt nd sl prt of the villi (Fig. 4E) nd were signifintly higher t dy 26 thn t irth (Fig. 4C), with tendeny to the highest numer in ENT pigs. However, the density ws similr etween neworn nd 26-dyold ENT nd TPN pigs (Fig. 4D). Gut permeility nd hexose sorptive pity. Figure 5A shows the urine ltulose/mnnitol rtio s mesure for the overll gut lekiness. Men vlues for permeility inresed fter irth in oth term nd preterm nimls with the highest men vlues in preterm pigs (P.12 t 26 dys). Five dys of ENT tended to lower the permeility in preterm nimls (P.5), nd susequent milk feeding for 21 dys ws not le to derese the lekiness of the preterm gut down to the level in orresponding term nimls. Lrge vritions in the dt set potentilly msked n overll effet of premturity (P.11) nd diet intervention (P.18). Plsm gltose inrement t 2 min in response to n orl olus of gltose ws mesured s mrker of hexose sorptive pity. The

6 A GLP-2 C Millions of L-ells G GLP-2 (pmol/l) 1 5 d ed d e numer of L-ells (millions) B GIP (pmol/l) GIP D L-ells density (numer *1/ muos volume) L-ell density E Age (d): ENT TPN ENT TPN ENTTPN ENT TPN ENT TPN Age (d): d d 26 TPN d 26 ENT Fig. 4. Plsm glugon-like peptide 2 (GLP-2, A) nd gluose-dependent insulin-tropi peptide (GIP, B) levels, numer of intestinl L ells (C), nd density of intestinl L ells (D nd E; rrows indite the GLP-1 stined ells) in -, 5-, nd 26-dy-old preterm or term pigs fed totl prenterl nutrition (TPN) or supplemented with enterl nutrition (ENT) for the first 5 dys (mens SE). Columns within the preterm or term groups not shring the sme letter re signifintly different (P.5). Signifint differene (P.1) etween neworn nd 26-dy-old preterm pigs, s nlyzed ross diets (ENT, TPN). ENT tretment tended to inrese vlues for oth delivery groups within the first week, ut the differenes were not signifint (P.16.18). By 4 wk, the term nimls hd inresed hexose sorptive pity, reltive to preterm pigs (P.1, Fig. 5B). Ex vivo digestive enzyme tivities. Ativity of six different rush order enzymes were nlyzed for preterm nd term pigs t ll three ges nd ross three intestinl regions (Fig. 6, A F). Previous studies show tht these enzyme tivities re diet dependent nd region speifi nd show differentil regultion y pre- nd postntl ge. Surse tivity (resulting from the surse-isomltse enzyme) nd mltse tivity (resulting from two enzymes, surse-isomltse nd mltsegluomylse) were low t irth nd there were no differenes etween preterm nd term pigs (left, Figs. 6A nd 5B). These enzyme tivities mtured with dvning postntl ge in oth delivery groups, espeilly in the proximl nd middle intestinl regions, ut preterm pigs were persistently ssoited with lower tivities. In the proximl intestine of 5-dy-old preterm nimls, surse nd mltse tivities were less thn 15% of the vlues in term nimls (Fig. 6, A nd B, middle). At this time, ENT slightly lowered the proximl surse nd mltse tivities in term pigs, reltive to vlues for TPN pigs (P.5), without ny effets for preterm pigs. At 26 dys, these enzyme tivities showed lrge inreses in oth proximl nd middle regions ut remined mrkedly lowered in preterm vs. term nimls (less thn 3%, right, Fig. 6, A nd B). At 26 dys, ENT tretment for 5 dys fter irth inresed

7 G556 A Permeility Urine L/M rtio A Surse (U/g) B Dy Dy 5 Dy 26 / ENT TPN ENT TPN / / / B Plsm gltose inrement (µmol/l) Gluose sorption ** Mltse (U/g) C Ltse (U/g) / / / / / / / / / / / ENT TPN ENT TPN ENT TPN ENTTPN Age (d): Fig. 5. Intestinl permeility [A, urinry ltulose/mnnitol (L/M) rtio] nd hexose sorptive pity (B, gltose inrement fter gltose olus) in -, 5-, nd 26-dy-old preterm or term pigs fed totl prenterl nutrition (TPN) or supplemented with enterl nutrition (ENT) for the first 5 dys (mens SE). Columns within the preterm or term groups not shring the sme letter re signifintly different (P.5). **Signifint differene (P.1) etween preterm nd term piglets for 26-dy-old pigs, nlyzed ross diets (ENT, TPN). the surse tivity in the proximl region for term pigs nd in the middle intestine for preterm pigs (oth P.5). In ontrst to surse nd mltse tivities, the tivity of ltse ws high t irth, ut more in term vs. preterm pigs, nd espeilly in the proximl nd middle regions (Fig. 6C, left). Ltse tivity deresed mrkedly in the postntl period of term pigs ut remined stle in preterm pigs (Fig. 6C, right, 26 dys). The ENT tretment lowered the proximl ltse tivity for oth preterm nd term pigs on dy 5 (P.1), ut this effet disppered y dy 26. Ativity of ll three peptidse enzymes inresed with postntl ge (P.1), espeilly fter dy 5, nd ws most pronouned in the middle nd distl intestine (Fig. 6, D F; 1% inreses from 5 to 26 dys of ge). The tivity of ApN on dy 5 ws inresed y ENT vs. TPN tretment in these intestinl regions (P.1), with no differenes etween preterm nd term pigs. On dy 26, the highest ApN tivity ws found in ENT preterm pigs nd the lowest vlues in term pigs fed only TPN for the first 5 dys (P.5, Fig. 6D, right). Ativity of ApA (Fig. 6E) ws lowered in preterm vs. term pigs for ll three postntl ge groups (P.1), mostly for neworn pigs ( 5%, P.1). At 5 dys, ut not t 26 dys, the ENT tretment inresed the ApA tivity (P D ApN (U/g) E ApA (U/g) F DPPIV (U/g) Prox Mid Dist / / // Prox / / / / / / / / / / Mid Dist Prox Mid Dist Fig. 6. Ativity of surse (A), mltse (B), ltse (C), minopeptidse N (ApN, D), minopeptidse A (ApA, E) nd dipeptidylpeptidse-4 (DPPIV, F) in -, 5-, nd 26-dy-old preterm or term pigs fed totl prenterl nutrition (TPN) or supplemented with enterl nutrition (ENT) for the first 5 dys (mens SE). In the figure, men vlues for eh intestinl region [proximl, middle, distl (dist)] nd ge fter irth (, 5, 26 dys) re different when they do not shre the sme supersript letter (,, ; dys, P.5). / /

8 .1), nd most for the preterm pigs. Ativity of DPP4 (Fig. 6F) ws signifintly lowered in preterm vs. term pigs t 5 nd 26 dys. At 5 dys, ENT inresed the DPP4 tivity for oth preterm nd term pigs, espeilly in the distl region (P.5). Aross ll the enzymes t 26 dys, ENT tretment for the first 5 dys ws ssoited with similr or higher men tivity of dishridses in the proximl nd middle intestine (Fig. 6, A C) nd similr or higher tivity of peptidses in the middle nd distl intestine (Fig. 6, D nd E). DISCUSSION Severl dedes of oservtionl studies in preterm infnts hve provided vlule informtion out the funtionl dpttion of mny orgns (e.g., lungs, liver, gut, nd rin) to preterm irth. Regrdless, studies in preterm infnts do not provide ny knowledge out the timing, extent, nd mehnisms of postntl orgn dpttion, nd ny diret omprison to dpttion in term infnts is usully not possile. It is importnt to understnd the onsequenes of preterm irth nd how est to llevite the short- nd long-term defiits in orgn funtion in preterm neontes. The preterm pig is the only niml model of preterm infnts tht not only shows the ommon respirtory nd metoli defiits of preterm irth ut lso spontneously develops mny of the immedite GI prolems of premturity, e.g., nutrient mldigestion, dysmotility, nd high sensitivity to NEC (43). Here, we show how intestinl struture nd funtion develop eyond the immedite neontl period in preterm pigs nd how erly introdution of enterl feeding my influene this development. We show tht the intestinl morphology of the immture intestine dpts surprisingly well nd eomes similr to tht in term pigs within 4 wk fter irth. In ontrst, digestive funtions suh s surse nd mltse tivities, rrier funtion, nd hexose sorptive pity remin ompromised t this ge. Erly nd grdul introdution of enterl milk (ENT tretment) enefited mny of the struturl nd funtionl indexes within the first week, espeilly in preterm pigs, ut most effets of ENT disppered y 4 wk. We onlude tht the preterm intestine dpts rpidly postntlly in pigs, prtly filitted y erly enterl food introdution, ut still, some developmentlly dependent digestive funtions remin immture until t lest 4 wk. It is not possile to sy extly wht this postntl ge in preterm pigs orresponds to in preterm infnts. On the other hnd, preterm pigs re likely to e more physiologilly mture t 4 wk thn preterm infnts, lso regrding the GI trt, s pigs would normlly enter into the wening trnsition period t this time. Together with lowered ody weight t irth, preterm pigs lso showed lowered intestine-to-ody weight rtio during the first week fter irth. During the following 3 wk, weight gin ws lowered in preterm pigs, despite identil rering nd feeding onditions, proly in prt explined y lowered nutrient digestive funtion, s indited y more dys with dirrhe. Although there ws persistent redution in villus heights in preterm pigs, there ws no overll redution in intestinl mss eyond the first week, nd our detiled stereologil mesurements long the entire smll intestine onfirmed tht totl intestinl muos volume ws similrly responsive in preterm nd term pigs to enterl food introdution nd dvning postntl ge. In ft, the postntl inrese in G557 reltive intestinl mss t 4 wk ws gretest in preterm pigs ( 2% from irth) nd this impressive trophi response my depend not only on postntl ge nd enterl food stimultion, ut lso on the speifi effet of enteroendorine signls suh s GLP-2. The GLP-2 relese in preterm pigs ws similr to, or even higher thn tht in term pigs (26 dys), despite tht L ell density per muos volume remined onstnt from irth to 4 wk in the preterm pigs nd etween ENT nd TPN fed nimls. Postntlly, the numer of L ells losely followed the inrese in muosl mss, despite tht other ell mrkers, suh s the high density of vuolted enteroytes, doumented tht the intestine ws indeed immture within the first week fter preterm irth. With inresing ge, higher rypt depths were oserved in oth preterm nd term nimls. Although prolifertion nd poptosis were not quntified in this study, this ould potentilly reflet inresed enteroyte turnover with dvning postntl ge (1). Despite tht mss nd volume of the muos grew similrly in preterm nd term pigs from irth to 4 wk, our study showed tht severl spets of muosl funtion filed to dpt nd showed more persistent developmentl dely. Consistent with inditions from preterm infnts (46), intestinl ltose digestive pity nd the sorption of gluose nd gltose y the sodium-oupled gluose trnsporter 1 (SGLT-1) were lowered in 26-dy-old preterm pigs, lthough ltse tivity (y the ltse-phloridzin hydrolse enzyme) ws most redued t irth (to 5% of vlues in term pigs). When surse nd mltse enzymti tivities mtured fter irth, vlues in 26-dy-old preterm pigs rehed only 3% of those in orresponding term pigs, nd vlues were similr to those in 5-dy-old term pigs. This mrked developmentl dely t 26 dys ws more thn expeted from the 12-dy differene in gesttionl ge t irth nd ws the most pronouned dely in intestinl funtion tht we oserved in preterm pigs. Although digestion of surose- nd mltose-ontining supplementry foods is of limited importne for preterm pigs nd infnts t this ge, the lking postntl dpttion is importnt, sine these enzymes re highly developmentlly dependent nd they re often used s key intestinl mturtion mrkers ross mny speies (39). Our results show tht the developmentl expression of these enzymes is reltively independent of environmentl ftors, suh s diet (whih ws the sme for preterm nd term pigs), nd tht intrinsi mehnisms relted to ontogeneti ge nd geneti ontrol ould e more importnt. We reently doumented tht oth preterm irth nd enterl food introdution indue epigeneti effets on some immune-relted genes in the immture pig intestine (19, 53). It will e importnt to know whether preterm irth lso indues epigeneti modifitions to the surse-isomltse nd mltsegluomylse genes euse this my help to explin why their orresponding enzyme tivities were ffeted more long term wheres other gene funtions tended to dpt more rpidly fter preterm irth. Enterl feeding promotes intestinl mturtion nd growth fter irth (39), ut speifilly for preterm neontes it is importnt to introdue enterl feeds grdully to minimize the risk of NEC (12, 52). We therefore used n erly nd slow linilly relevnt feeding regimen for preterm pigs, ompring ENT with TPN pigs. We used ovine olostrum euse this enterl diet indues intestinl mturtion nd NEC protetion in preterm pigs (39) nd hs reently een tested s the

9 G558 first enterl diet for preterm infnts (28). The intestinl trophi effets of the ENT tretment for the first 5 dys generlly disppered y 26 dys in oth preterm nd term pigs. Nevertheless, severl lines of evidene suggest tht erly nd slow introdution of trophi milk diets my e enefiil for preterm infnts more long term (15). In our study, the ENT diet did not ffet villus heights nd muos volumes t dy 5 ut it inresed reltive intestinl weight, peptidse tivities, nd the endorine GLP-2 nd GIP relese, nd it tended to improve muosl rrier nd gluose sorptive pity. Interestingly, these effets were s high, or higher, in preterm vs. term pigs, nd this my help dpttion of the immture intestine to lter full enterl feeding. We did not in this study mke detiled reord of feeding intolerne nd intestinl dysmotility, ut, like in preterm infnts, gstri residuls nd vomiting were osionlly oserved in preterm pigs during the first 3 wk fter irth. During this ritil period, intestinl permeility ws lso higher nd our results indite tht even smll mounts of enterl diet help to mture the intestinl rrier. In preterm infnts, intestinl permeility is high during the first weeks fter irth followed y derese, proly in prt medited y inresing milk intke (46) nd GLP-2 relese (35). Although suh trophi effets of the first enterl feeds help to inrese muosl growth nd mture some intestinl funtions, others my remin ompromised for longer period, s we demonstrted in this study on piglets for surse nd mltse tivities in the proximl nd middle intestine. Exogenous GLP-2 dministrtion stimultes intestinl growth nd dishridse tivities in TPN-fed preterm pigs with or without intestinl resetion (37, 49, 51). piglets normlly show inresed sl irulting levels of GLP-2, ompred with pigs orn t term, inditing role for GLP-2 in the mturtionl proess round irth (38). In the present study, preterm pigs displyed low surse nd mltse tivities despite reltively high postntl GLP-2 levels, nd the ENT tretment tended to ffet GIP more thn GLP-2 relese within the first 5 dys wheres gstrin ws little ffeted. Proly the smll volumes of enterl milk exposed minly the proximl prt of the intestine, nd therey the GIP-produing K ells, wheres the gstrin-produing stomh G ells nd GLP-2 sereting L ells in the distl smll intestine were less ffeted. Suh enteroendorine ells my e present s fixed proportion of the totl numer of ells in the muos, regrdless of time of irth nd inresing muosl mss during the postntl period. Hene, we oserved tht the L ell density ws onstnt from irth to 26 dys in preterm pigs nd we did therefore not to investigte this prmeter in the remining groups. The enteroendorine ells hve reltively slow ell turnover [round 2 dys (11)], mking their totl numer less sensitive to environmentl stimuli. Thus, from n enteroendorine perspetive, preterm pigs ppered reltively mture lredy t irth. As prt of perintl mturtion, the smll intestine undergoes hnges in its regionl distriution of speifi digestive funtions (42). Consistent with this, the proximl-to-distl derese in villus height nd ltse tivities within the first week, nd of surse nd mltse tivities t 4 wk, were muh less pronouned in preterm vs. term pigs. Reltive to dishridses, peptidse tivities showed less distint ge-relted postntl mturtion lthough they were more sensitive to enterl food stimultion. Aordingly, in oth preterm nd term pigs, peptidse tivities were found minly in the middle nd distl intestine t 26 dys, nd ENT nutrition minly stimulted peptidse tivities in these regions on dy 5. Previous studies in pre- nd postntl pigs onfirm tht the intestine mtures in proximl to distl diretion nd tht the enzyme tivities re most sensitive to ge nd hormonl stimuli (e.g., ortisol, GLP-2) proximlly, wheres enterl milk diets ffet enzyme tivities (e.g., peptidses) most in the distl prts (39, 42). We onlude tht postntl dpttion of intestinl struture nd funtion differs mong different intestinl regions nd tkes ple in omplex interply mong ontogeneti ge, gesttionl ge t irth, nd diet. Although intestinl morphology nd struture dpt rpidly within the first 4 wk in preterm pigs, seleted gut funtions remin ompromised nd elow the levels in orresponding term pigs. Even smll mounts of enterl milk feeds improve intestinl morphology nd funtion within the first week fter irth ut hve limited effets 3 4 wk lter. Perspetives It is importnt to know whether the pprent defiits oserved following preterm irth re temporry or more long lsting, nd whether they re ffeted y the erly enterl diet. A lsting funtionl defiit of the intestine my ontriute to the ommonly oserved extruterine growth restrition in preterm infnts (14). Our study suggests tht the immture intestinl morphology in neworn preterm pigs is lrgely normlized within the first 4 wk fter irth, while severl spets of intestinl funtion remin immture. Cution is required when extrpolting intestinl premturity in pigs t 9% gesttion to the highly vrile group of preterm infnts orn t 6 9% gesttion, espeilly sine the development of eh orgn system is temporlly different etween humns nd pigs. Although pigs delivered t 9% gesttion show severe GI, respirtory, nd metoli immturities, some other funtions (e.g., neurodevelopment) pper more mture in preterm pigs thn in most preterm infnts (14). Regrdless, it is importnt to identify the GI funtions tht show lsting defiits following preterm irth in eh speies euse this my help to design optiml supportive nd preventive re. GRANTS This reserh ws supported y the Dnish Counil for Strtegi Reserh (NEOMUNE reserh enter) nd Nutrii Reserh nd ARLA Food Ingredients. DISCLOSURES No onflits of interest, finnil or otherwise, re delred y the uthor(s). AUTHOR CONTRIBUTIONS C.F.H., T.T., A.D.A., J.J.H., B.H., L.H., nd L.L. performed experiments; C.F.H., T.T., nd L.L. nlyzed dt; C.F.H., T.T., L.L., nd P.T.S. interpreted results of experiments; C.F.H. prepred figures; C.F.H. nd P.T.S. drfted mnusript; C.F.H., T.T., A.D.A., J.J.H., B.H., L.L., J.J., nd P.T.S. edited nd revised mnusript; C.F.H., T.T., A.D.A., nd P.T.S. pproved finl version of mnusript; T.T. nd P.T.S. oneption nd design of reserh. REFERENCES 1. Amin H, Holst JJ, Hrtmnn B, Wlle L, Wright J, Siglet DL. Funtionl ontogeny of the proglugon-derived peptide xis in the premture humn neonte. Peditris 121: e18 e186, Andersen AD, Sngild PT, Munh SL, vn der Beek EM, Renes IB, Vn Ginneken C, Greisen GO, Thymnn T. Delyed growth, motor

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